A protocol for measuring the activity of protein kinase C-delta in murine bone-marrow-derived dendritic cells

Abstract

Summary: Protein kinase C-δ (PKC-δ) is a key enzyme controlling growth, differentiation, and apoptosis in various cells, including immune cells. Here, we present a protocol to determine PKC-δ activation in response to increased membrane-bound diacylglycerol or phorbol-12-myristate-13-acetate treatment in murine bone-marrow-derived dendritic cells. We describe steps for dendritic cell differentiation, the isolation of plasma membrane lipids, and the quantification of diacylglycerol. We then detail procedures for measuring PKC-δ kinase activity by in vitro assay, indirect immunofluorescence, and western blotting experiments.For complete details on the use and execution of this protocol, please refer to Parsons et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell culture
• Cell Membrane
• Cell-based Assays
• Cell separation/fractionation
• Genetics
• Immunology